In cartilage, reactive oxygen species (ROS) signaling plays significant role in regulating chondrocyte proliferation, differentiation and maturation. To assure proper cellular function ROS is balanced by production of antioxidants;as unregulated ROS can cause dysfunction in gene expression, transcription factor signaling, and cell cycle. In aging, systemic disease, environmental toxin exposure, injury and inflammation, ROS production can overwhelm the antioxidant capacity triggering aberrant signaling leading to cell death, matrix degradation and pathological damage in both forming and permanent cartilages. Apoptosis signal-regulating kinase 1 (ASK1) a MAP kinase kinase kinase, is a well characterized protein implicated in pathological ROS signaling. In our preliminary data, we show Ask1 is present in growth plate and articular chondrocytes. We also show that hypoxia inducible factor 2 (HIF-2) is a transcriptional regulator controlling antioxidant expression in chondrocytes. Taken together, we propose that in cartilage the oxidative status of the cell is controlled by expression of HIF-2;and activation of Ask1 is a functional measurement of oxidative stress. We will investigate the relationship between HIF-2 controlled expression of antioxidants and activation of Ask1 by ROS. By manipulating the expression of these two molecules we will explore the normal and pathological role of ROS in chondrocyte differentiation and stasis. In Specific Aim 1, using siRNA to silence Ask1 gene expression we will investigate the mechanism of action of Ask1 as an indicator of oxidative stress in chondrocytes and its role in ROS induced cartilage pathology. In Specific Aim 2, we will investigate the expression of HIF-2 in controlling cellular oxidative status by antioxidant regulation. By manipulating the expression HIF-2, using siRNA to silence HIF-2 gene expression and a hypoxia independent plasmid for overexpression, we will determine cellular oxidative status, ROS mediated activation of Ask1, chondrocyte catabolic activity and survival. By carefully analyzing the signaling mechanisms altered by oxidative stress in chondrocytes, our proposed studies will have an enormous impact on understanding molecular mechanisms of oxidative stress, and provide important information for developing novel targets for therapeutic intervention. Public Health Relevance: This proposal will examine the relationship between environmental oxidants and antioxidant expression in cartilage cells;how this balance affects cartilage cell function and cartilage degeneration. Studies will include analysis of tissue and isolated cells from mice with impaired or improved antioxidant function and human osteoarthris tissue to determine these relationships. We will perform analyses to determine if genes that alter cartilage cell function are increased leading to the development of cartilage dysfunction.